Summary of Study ST002422

This data is available at the NIH Common Fund's National Metabolomics Data Repository (NMDR) website, the Metabolomics Workbench, https://www.metabolomicsworkbench.org, where it has been assigned Project ID PR001559. The data can be accessed directly via it's Project DOI: 10.21228/M85X3W This work is supported by NIH grant, U2C- DK119886.

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This study contains a large results data set and is not available in the mwTab file. It is only available for download via FTP as data file(s) here.

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Study IDST002422
Study TitleUBXD8 lipidomics from whole cells (Part 2)
Study SummaryThe intimate association between the endoplasmic reticulum (ER) and mitochondrial membranes at ER-mitochondria contact sites (ERMCS) serves as a platform for several critical cellular processes, in particular lipid synthesis. Enzymes involved in lipid biosynthesis are enriched at contacts and membrane lipid composition at contacts is distinct relative to surrounding membranes. How contacts are remodeled and the subsequent biological consequences of altered contacts such as perturbed lipid metabolism remains poorly understood. Here we investigate if the ER-tethered ubiquitin-X domain adaptor 8 (UBXD8) regulates the lipids found in mitochondria-associated membranes (MAM). LC-MS/MS lipidomics found significant changes in distinct lipid species in the MAM fraction of UBXD8 knockout cells. Our results suggest that lipids in MAM are regulated by UBXD8.
Institute
University of Arizona
DepartmentImmunobiology
LaboratoryPurdy Lab
Last NamePurdy
First NameJohn
AddressPO Box 245221, Tucson, Arizona, 85724, USA
Emailpurdylab@gmail.com
Phone520-626-4371
Submit Date2023-01-01
Raw Data AvailableYes
Raw Data File Type(s)mzXML
Analysis Type DetailLC-MS
Release Date2023-01-16
Release Version1
John Purdy John Purdy
https://dx.doi.org/10.21228/M85X3W
ftp://www.metabolomicsworkbench.org/Studies/ application/zip

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Project:

Project ID:PR001559
Project DOI:doi: 10.21228/M85X3W
Project Title:UBXD8 lipidomics from whole cells
Project Summary:The intimate association between the endoplasmic reticulum (ER) and mitochondrial membranes at ER-mitochondria contact sites (ERMCS) serves as a platform for several critical cellular processes, in particular lipid synthesis. Enzymes involved in lipid biosynthesis are enriched at contacts and membrane lipid composition at contacts is distinct relative to surrounding membranes. How contacts are remodeled and the subsequent biological consequences of altered contacts such as perturbed lipid metabolism remains poorly understood. Here we investigate if the ER-tethered ubiquitin-X domain adaptor 8 (UBXD8) regulates the lipidome of cells. LC-MS/MS lipidomics found significant changes in distinct lipid species in UBXD8 knockout cells, in particular in saturated or mono-unsaturated lipid species. Perturbation of contacts and inherent lipid synthesis is emerging as a hallmark in a variety of human disorders such as neurodegeneration. Our results suggest that contacts are exquisitely sensitive to alterations to membrane lipid composition and saturation in a manner that is dependent on UBXD8.
Institute:University of Arizona
Department:Immunobiology
Laboratory:Purdy Lab
Last Name:Purdy
First Name:John
Address:PO Box 245221, Tucson, Arizona, 85724, USA
Email:purdylab@gmail.com
Phone:520-626-4371
Funding Source:NIH R01 AI162671
Contributors:Rakesh Ganji, Joao A. Paulo, Yuecheng Xi, Ian Kline, Jiang Zhu, Christoph S. Clemen, Conrad C. Weihl, John G. Purdy, Steve P. Gygi, and Malavika Raman

Subject:

Subject ID:SU002511
Subject Type:Cultured cells
Subject Species:Homo sapiens
Taxonomy ID:9606
Cell Strain Details:HEK293T

Factors:

Subject type: Cultured cells; Subject species: Homo sapiens (Factor headings shown in green)

mb_sample_id local_sample_id Description inj vol
SA24237920220820_tufts_MAMfrac_neg_rep8_KO_10ulUBXD8 Knockout 10
SA24238020220508_neg_KO_rep4_10ulUBXD8 Knockout 10
SA24238120220508_neg_KO_rep3_10ulUBXD8 Knockout 10
SA24238220220508_neg_KO_rep1_10ulUBXD8 Knockout 10
SA24238320220820_tufts_MAMfrac_neg_rep8_KO_15ulUBXD8 Knockout 15
SA24238420220508_pos_KO_rep3_16ulUBXD8 Knockout 16
SA24238520220820_tufts_MAMfrac_pos_rep8_KO_16ulUBXD8 Knockout 16
SA24238620220508_pos_KO_rep4_16ulUBXD8 Knockout 16
SA24238720220508_pos_KO_rep1_16ulUBXD8 Knockout 16
SA24238820220820_tufts_MAMfrac_pos_rep8_KO_4ulUBXD8 Knockout 4
SA24238920220508_pos_KO_rep3_4ulUBXD8 Knockout 4
SA24239020220508_pos_KO_rep1_4ulUBXD8 Knockout 4
SA24239120220508_pos_KO_rep4_4ulUBXD8 Knockout 4
SA24239220220820_tufts_MAMfrac_neg_rep8_KO_5ulUBXD8 Knockout 5
SA24239320220508_pos_KO_rep1_8ulUBXD8 Knockout 8
SA24239420220508_pos_KO_rep4_8ulUBXD8 Knockout 8
SA24239520220820_tufts_MAMfrac_pos_rep8_KO_8ulUBXD8 Knockout 8
SA24239620220508_pos_KO_rep3_8ulUBXD8 Knockout 8
SA24239720220508_neg_WT_rep3_10ulWild-type 10
SA24239820220508_neg_WT_rep4_10ulWild-type 10
SA24239920220508_neg_WT_rep1_10ulWild-type 10
SA24240020220820_tufts_MAMfrac_neg_rep8_WT_10ulWild-type 10
SA24240120220820_tufts_MAMfrac_neg_rep8_WT_15ulWild-type 15
SA24240220220508_pos_WT_rep3_16ulWild-type 16
SA24240320220508_pos_WT_rep1_16ulWild-type 16
SA24240420220508_pos_WT_rep4_16ulWild-type 16
SA24240520220820_tufts_MAMfrac_pos_rep8_WT_16ulWild-type 16
SA24240620220820_tufts_MAMfrac_pos_rep8_WT_4ulWild-type 4
SA24240720220508_pos_WT_rep3_4ulWild-type 4
SA24240820220508_pos_WT_rep4_4ulWild-type 4
SA24240920220508_pos_WT_rep1_4ulWild-type 4
SA24241020220820_tufts_MAMfrac_neg_rep8_WT_5ulWild-type 5
SA24241120220508_pos_WT_rep1_8ulWild-type 8
SA24241220220508_pos_WT_rep3_8ulWild-type 8
SA24241320220508_pos_WT_rep4_8ulWild-type 8
SA24241420220820_tufts_MAMfrac_pos_rep8_WT_8ulWild-type 8
Showing results 1 to 36 of 36

Collection:

Collection ID:CO002504
Collection Summary:Cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM), supplemented with 10% fetal bovine serum (FBS) and 100 units/mL penicillin and streptomycin. Cells were maintained in a humidified, 5 % CO2 atmosphere at 37°C. The CRISPR-Cas9 gene editing system was used to generate UBXD8 knockout cell lines in HEK293T cells. Cells were seeded into four 150 mm TC dishes. Cells were lysed in Homogenization buffer (225 mM mannitol, 75 mM sucrose, and 30 mM Tris-Cl, pH 7.4) using a Dounce homogenizer. The lysate was centrifuged three times at 600xg for 5 minutes to remove unlysed cells and nuclei resulting in post-nuclear supernatants (PNS). The cleared lysate was centrifuged at 7000xg to separate crude mitochondrial pellet and supernatant containing microsomes. The supernatant was cleared by centrifugation at 20,000xg for 30 minutes followed by microsome isolation using high-speed centrifugation at 100,000xg for 1 hour. The crude mitochondrial pellet was washed twice in homogenization buffer containing 0.1 mM EGTA at 7000xg and 10,000xg for 10 minutes. MAMs were isolated from crude mitochondria using 30 % Percoll gradient centrifugation at 95,000xg for 1 hr in a swinging-bucket rotor. The banded MAM fraction was washed once with phosphate-buffered saline (PBS) before lysing in lysis buffer (50 mM Tris-Cl, pH 7.2, 150 mM NaCl). The pure mitochondrial fractions were resuspended and washed in mitochondrial resuspension buffer (250 mM mannitol, 0.5 mM EGTA, 5 mM HEPES pH7.4). Protein concentrations for both soluble and pellet fractions were determined by DC protein assay kit (Biorad). For lipidomics of MAMs, the final banded MAM fraction was washed twice with liquid chromatography-mass spectrometry (LC-MS) grade PBS and the final MAM pellet was resuspended in LC-MS grade PBS. Next, 1mL of cold 50% methanol was added and the MAMS were transferred to glass vials. Chloroform was added (0.5mL) and the mixture was gently vortexed and centrifuged at 1,000x g for 5 min at 4˚C. Lipids were transferred to a clean glass vial using a glass Hamilton syringe. Lipids were extracted twice using chloroform prior to being dried under nitrogen gas. Samples were normalized according to protein concentration. Extracted lipids were resuspended in a 1:1:1 solution of methanol:chloroform:isopropanol prior to mass spectrometry (MS) analysis.
Sample Type:Epithelial cells

Treatment:

Treatment ID:TR002523
Treatment Summary:Wild-type vs UBXD8 Knockout

Sample Preparation:

Sampleprep ID:SP002517
Sampleprep Summary:Lipids were isolated from collected cultured cells. Cells were washed with PBS, treated with cold 50% methanol (1mL) and transferred to glass vials. Next, chloroform (0.5mL) was added and samples were gently vortexed and centrifuged at 1,000x g for 5 min at 4˚C. Lipids were transferred to a clean glass vial using a glass Hamilton syringe. Lipids were extracted twice using chloroform prior to being dried under nitrogen gas. Samples were normalized according to protein concentration when resuspended in a 1:1:1 solution of methanol:chloroform:isopropanol prior to mass spectrometry (MS) analysis. To assist quantification various volumes were injected for MS (4, 8, and 16ul for positive mode analysis and 5, 10, and 15ul for negative mode analysis).

Combined analysis:

Analysis ID AN003944 AN003945
Analysis type MS MS
Chromatography type Reversed phase Reversed phase
Chromatography system Thermo Vanquish Thermo Vanquish
Column Waters ACQUITY UPLC CSH C18 (150 x 2.1mm,1.7um) Waters ACQUITY UPLC CSH C18 (150 x 2.1mm,1.7um)
MS Type ESI ESI
MS instrument type Orbitrap Orbitrap
MS instrument name Thermo Q Exactive Plus Orbitrap Thermo Q Exactive Plus Orbitrap
Ion Mode POSITIVE NEGATIVE
Units peak area peak area

Chromatography:

Chromatography ID:CH002920
Chromatography Summary:Lipids were identified and quantitatively measured using ultra high-performance liquid-chromatography high-resolution tandem MS/MS (UHPLC-MS/MS) as recently described89,90. Separation of lipids was done by reverse-phase chromatography using a Waters ACQUITY CSH C18 column (150x2.1mm; 1.7um) at 60°C using a Vanquish UHPLC system (Thermo Scientific) and two solvents: solvent A (40:60 water-methanol plus 10mM ammonium formate and 0.1% formic acid) and solvent B (10:90 methanol-isopropanol plus 10mM ammonium formate and 0.1% formic acid). UHPLC was performed at a 0.25 ml per min flow rate for 30 min per sample, starting at 25% solvent B and ending at 100% solvent B as described. The column was washed and equilibrated between samples. Samples were run in a semi-random order where WT or UBXD8 KO samples were interspersed with blank samples. Each sample was repeatedly analyzed using various injection volumes (4, 8, 16ul for positive mode and 5, 10, and 15ul for negative mode).
Instrument Name:Thermo Vanquish
Column Name:Waters ACQUITY UPLC CSH C18 (150 x 2.1mm,1.7um)
Column Temperature:60
Flow Gradient:25% to 100%
Flow Rate:0.25mL per min
Solvent A:40% water; 60% methanol 10mM ammonium formate and 0.1% formic acid
Solvent B:10% methanol; 90% isopropanol 10mM ammonium formate and 0.1% formic acid
Chromatography Type:Reversed phase

MS:

MS ID:MS003680
Analysis ID:AN003944
Instrument Name:Thermo Q Exactive Plus Orbitrap
Instrument Type:Orbitrap
MS Type:ESI
MS Comments:Samples were run in a semi-random order where WT or UBXD8 KO samples were interspersed with blank samples. Lipids were ionized using a heated electrospray ionization (HESI) source and nitrogen gas and measured using a Q-Exactive Plus mass spectrometer operating at a MS1 resolution of either 70,000 or 140,000 and a MS2 resolution of 35,000. MS1 Spectra were collected over a mass range of 200 to 1,600 m/z with an automatic gain control (AGC) setting of 1e6 and transient times of 250 ms (70,000 resolution) or 520 ms (140,000 resolution). MS2 spectra were collected using a transient time of 120 ms and an AGC setting of 1e5. Each sample was analyzed using negative and positive ion modes. The mass analyzer was calibrated weekly. SPLASH LIPIDOMIX mass spectrometry standards (Avanti Polar Lipids) were used in determining extraction efficiencies and lipid quantitation. Lipids were identified and quantified using MAVEN, and El-MAVEN (Elucidata). UHPLC retention time, MS1 peaks, and MS2 fragments were used to identify lipids. Lipids were included if they were observed in 3-6 samples in both UBXD8 KO and WT cells. Missing values in a sample were not imputed. The following lipid classes were included in the analysis: cholesteryl esters (CE), diacylglycerol (DG), phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylglycerol (PG), phosphatidylinositol (PI), phosphatidylserine (PS), and triacylglycerol (TG). Guidelines from the Lipidomic Standards Initiative were followed for lipid species identification and quantification, including consideration of isotopic patterns resulting from naturally occurring 13C atoms and isomeric overlap. The following MS2 information was used to confirm each lipid species: PC fragment of 184.073 (positive mode) and tail identification using formic adduct (negative mode); PE fragment of 196.038 or the tail plus 197.046 (negative mode) and neutral loss (NL) of 141.019 (positive mode); PG fragment of 152.996 plus the identification of the FA tails (negative mode) and NL 189.04 of [M+NH4]+ adduct (positive mode); PI fragment of 241.012 (negative) and NL 277.056 of [M+NH4]+ adduct (positive mode); PS NL of 87.032 (negative); DG and TG by NL of FA tails (positive mode); and CE fragment of 369.352 or neutral loss of 368.35 (positive).
Ion Mode:POSITIVE
  
MS ID:MS003681
Analysis ID:AN003945
Instrument Name:Thermo Q Exactive Plus Orbitrap
Instrument Type:Orbitrap
MS Type:ESI
MS Comments:Samples were run in a semi-random order where WT or UBXD8 KO samples were interspersed with blank samples. Lipids were ionized using a heated electrospray ionization (HESI) source and nitrogen gas and measured using a Q-Exactive Plus mass spectrometer operating at a MS1 resolution of either 70,000 or 140,000 and a MS2 resolution of 35,000. MS1 Spectra were collected over a mass range of 200 to 1,600 m/z with an automatic gain control (AGC) setting of 1e6 and transient times of 250 ms (70,000 resolution) or 520 ms (140,000 resolution). MS2 spectra were collected using a transient time of 120 ms and an AGC setting of 1e5. Each sample was analyzed using negative and positive ion modes. The mass analyzer was calibrated weekly. SPLASH LIPIDOMIX mass spectrometry standards (Avanti Polar Lipids) were used in determining extraction efficiencies and lipid quantitation. Lipids were identified and quantified using MAVEN, and El-MAVEN (Elucidata). UHPLC retention time, MS1 peaks, and MS2 fragments were used to identify lipids. Lipids were included if they were observed in 3-6 samples in both UBXD8 KO and WT cells. Missing values in a sample were not imputed. The following lipid classes were included in the analysis: cholesteryl esters (CE), diacylglycerol (DG), phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylglycerol (PG), phosphatidylinositol (PI), phosphatidylserine (PS), and triacylglycerol (TG). Guidelines from the Lipidomic Standards Initiative were followed for lipid species identification and quantification, including consideration of isotopic patterns resulting from naturally occurring 13C atoms and isomeric overlap. The following MS2 information was used to confirm each lipid species: PC fragment of 184.073 (positive mode) and tail identification using formic adduct (negative mode); PE fragment of 196.038 or the tail plus 197.046 (negative mode) and neutral loss (NL) of 141.019 (positive mode); PG fragment of 152.996 plus the identification of the FA tails (negative mode) and NL 189.04 of [M+NH4]+ adduct (positive mode); PI fragment of 241.012 (negative) and NL 277.056 of [M+NH4]+ adduct (positive mode); PS NL of 87.032 (negative); DG and TG by NL of FA tails (positive mode); and CE fragment of 369.352 or neutral loss of 368.35 (positive).
Ion Mode:NEGATIVE
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